AU-rich Region Research Articles

Fabricating the AuPdPt Ternary Nanophase Diagram at 800 °C To Guide the Exploration of Optimal Catalyst for n-Hexane Oxidation

A good knowledge of nanophase diagrams is critical to understanding the performance of nanomaterials. Herein, we for the first time fabricate a AuPdPt ternary nanophase diagram at 800 °C by experimental means. The solid-state synthesis strategy enables a precise synthesis of AuPdPt trimetallic nanoparticles with a full range of compositions and desired size distributions. By studying the phase behavior of those TMNPs, we succeed in mapping out the boundary curve and tie lines. Interestingly, the AuPdPt nanophase boundary shifts obviously to the Au-rich region as compared to the case of the corresponding bulk diagram, likely due to the combination of size and composition effects. Besides, owing to the different solubility of Pd in Au-rich and Pt-rich matrix, some tie lines do not parallel the baseline. These observations greatly improve our basic understanding of nanophase diagrams and are expected to be valuable references for other systems. Moreover, the as-fabricated nanophase diagram is successfully ap...

Abstract 12532: The Tandem Zinc Finger mRNA Binding Protein, Tristetraprolin, is a Novel Regulator of Cardiac Fatty Acid Metabolism Through its Effect on PPARa

Introduction: Altered substrate utilization has been described in type II diabetes and heart failure (HF), but current therapies do not target the metabolic derangements in these disorders. Identification of novel pathways regulating cellular metabolism could facilitate the development of new therapies. Tristetraprolin (TTP) is a tandem zinc finger protein that binds to AU-rich regions in the 3’ untranslated region (UTR) of mRNA molecules and causes their degradation. Its expression is reduced in patients with diabetes, and its cellular level is regulated by mTOR, a key protein involved in cellular metabolism. Furthermore, the yeast homolog of TTP has been suggested to play a role in metabolism. Thus, we hypothesized that TTP regulates cardiac metabolism and plays a role in the development of HF through its effects on substrate utilization in the heart. Results: We first assessed the effects of TTP modulation on cellular substrate utilization, and found that TTP downregulation in cultured cardiomyocytes resulted in higher palmitate uptake and oxidation while its overexpression had the opposite effect. Since TTP regulates its targets at the mRNA level, we studied the mRNA levels of all genes involved in lipid metabolism, and found that only PPARα mRNA to be significantly increased with TTP downregulation. Furthermore, we demonstrated that TTP physically interacts with PPARα mRNA, and the activity of a luciferase reporter harboring full-length PPARα 3’UTR is increased with TTP downregulation. Additionally, PPARα mRNA is stabilized with TTP knockdown. We then studied the role of TTP in cardiac metabolism using mice with cardiac-specific TTP knockout. Although cardiac-specific TTP knockout mice had normal cardiac function at baseline, they displayed higher fatty acid utilization compared to wild type littermate control in an ex vivo heart perfusion setup. We also demonstrated a significantly higher TTP levels in failing human and mouse cardiac samples, suggesting that TTP levels are altered in HF. Conclusion: Our results demonstrate that TTP is a novel regulator of cardiac fatty acid metabolism through its effect on PPARα, and that its levels are increased in HF. Thus, modulation of TTP may be a viable therapeutic approach for HF.

Competing Repressive Factors Control Bone Morphogenetic Protein 2 (BMP2) in Mesenchymal Cells.

The amount, timing, and location of bone morphogenetic protein 2 (BMP2) synthesis influences the differentiation of pluripotent mesenchymal cells in embryos and adults. The BMP2 3'untranslated region (3'UTR) contains a highly conserved AU-rich element (ARE) embedded in a sequence that commonly represses gene expression in mesenchymal cells. Computational analyses indicate that this site also may bind several microRNAs (miRNAs). Although miRNAs frequently target AU-rich regions, this ARE is unusual because the miRNAs directly span the ARE. We began to characterize the factors that may regulate Bmp2 expression via this complex site. The activating protein HuR (Hu antigen R, ELAVL1, HGNC:3312) directly binds this ARE and can activate gene expression. An miRNA was demonstrated to reverse HuR-mediated activation. Mutational and RNA-interference evidence also supports an AUF1 (AU-factor-1, HNRNPD, HGNC:5036) contribution to the observed repressive activity of the 3'UTR in mesenchymal cells. A limited number of studies describe how miRNAs interact with ARE-binding proteins that bind adjacent sites. This study is among the first to describe protein/miRNA interactions at the same site.

Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster Ovary cells

Background: Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. Destabilization of dihydrofolate reductase (dhfr) selection marker by addition of AU-rich elements and murine ornithine decarboxylase PEST region was previously shown to improve the specific productivities of recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated novel combinations of engineered motifs for further selection marker attenuation to improve recombinant human alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency. Results: After a 2-step methotrexate (MTX) amplification to 50 nM that took less than 3 months, the expression vector with IRES point mutation and dhfr-PEST gave a maximum titer of 1.05 g/l with the top producer cell pool. Further MTX amplification to 300 nM MTX gave a maximum titer of 1.15 g/l. Relative transcript copy numbers and dhfr protein expression in the cell pools were also analysed to demonstrate that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, and that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in suspension serum free culture. Conclusions: Novel combinations of engineered motifs for further selection marker attenuation were studied to result in the highest reported recombinant protein titer to our knowledge in shake flask batch culture of stable mammalian cell pools at 1.15 g/l, highlighting applicability of expression vector optimization in generating high producing stable cells essential for recombinant protein therapeutics production. Our results also suggest that codon usage of the selection marker should be considered for applications that may involve gene amplification and serum free suspension culture, since the overall codon usage and thus the general expression and regulation of host cell proteins may be affected in the surviving cells.

Genetic deletion of AU-rich region in the tristetraprolin (TTP) transcript increases TTP stability and expression and provides protection against inflammatory diseases (INM1P.433)

Abstract Tristetraprolin (TTP) is an mRNA binding protein that binds to AU-rich elements (AREs) in the 3’-untranslated regions (3’UTR) of specific mRNAs, such as that encoding tumor necrosis factor (TNF), and increases their rate of mRNA turnover. TTP mRNA also contains AREs within its own 3’UTR that are partly responsible for its own rapid turnover. In the present study, we generated a novel TTP mouse line (TTPΔARE) with a 136 base deletion in the 3’UTR AU-rich region of TTP mRNA, and studied the effect of this genetic modification on the levels of TTP mRNA and protein, and susceptibility of the mice to experimentally-induced inflammatory conditions. TTP mRNA stability was found to be enhanced in TTPΔARE bone marrow-derived macrophages (BMDMs) and mouse embryonic fibroblasts (MEFs). TTP protein expression was increased, both in the basal state and upon LPS stimulation, in BMDMs, and in selected mouse tissues. LPS-induced expression of TTP target mRNAs, particularly Tnf and Il-1β, was significantly reduced in TTPΔARE BMDMs. Most importantly, TTPΔARE mice were almost completely protected from collagen antibody-induced arthritis, and exhibited significantly reduced inflammation in an imiquimod-induced model of psoriasis. These data suggest that increased levels of TTP can protect against some experimentally induced inflammatory diseases. They also raise the possibility that increasing TTP levels in man might be an attractive therapeutic approach in some inflammatory conditions.

Post‐transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich regions in target mRNAs, leading to their deadenylation and decay. Family members in Saccharomyces cerevisiae influence iron metabolism, whereas the single protein expressed in Schizosaccharomyces pombe, Zfs1, regulates cell-cell interactions. In the human pathogen Candida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts. Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3'UTR, compared with only 3 of 56 (5%) down-regulated transcripts. The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole, perhaps because of increased expression of the potential target transcript encoded by HIS3. Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade, while the predicted Zfs1 binding sites in these mRNAs often 'disappeared' with increasing evolutionary distance from the parental species. C. albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity, and Zfs1 was associated with target transcripts after co-immunoprecipitation. Thus, the biochemical activities of these proteins in fungi are highly conserved, but Zfs1-like proteins may target different transcripts in each species.

Tristetraprolin is a novel regulator of BDNF

Brain-derived neurotrophic factor (BDNF) regulates multiple biological processes ranging from central nervous system development and function to neuroinflammation and myogenic differentiation and repair. While coordination of BDNF levels is central in determining the biological outcome, mechanisms involved in controlling BDNF levels are not fully understood. Here we find that both short (BDNF-S) and long (BDNF-L) BDNF 3’UTR isoforms contain conserved adenylate- and uridylate rich elements (AREs) that may serve as binding sites for RNA-binding proteins (ARE-BPs). We demonstrate that ARE-BPs tristetraprolin (TTP) and its family members butyrate response factor 1 (BRF1) and 2 (BRF2) negatively regulate expression from both BDNF-S and BDNF-L containing transcripts in several cell-lines and that interaction between TTP and AU-rich region in proximal 5’ end of BDNF 3’UTR is direct. In line with the above, endogenous BDNF mRNA co-immunoprecipitates with endogenous TTP in differentiated mouse myoblast C2C12 cells and TTP overexpression destabilizes BDNF-S containing transcript. Finally, RNAi-mediated knock-down of TTP increases the levels of endogenous BDNF protein in C2C12 cells. Our findings uncover TTP as a novel regulator of BDNF assisting future studies in different physiological and pathological contexts.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-502) contains supplementary material, which is available to authorized users.

The RNA-binding protein HuR is a posttranscriptional on switch for CD4+ Th2 and Th17 differentiation (LYM3P.730)

Abstract Posttranscriptional control by RNA binding proteins (RBPs) in CD4+ T cells is poorly understood. The RBP, HuR, regulates IL-4, IL-13 and GATA-3, by binding to AU-rich (ARE) regions in their 3’ UTRs. We hypothesized HuR is regulating CD4+ Th2 differentiation by controlling mRNA stability. We previously showed that HuR over-expression results in increased Th2 differentiation and Th2 cytokines. We generated a new model to study Th2 differentiation, distal Lck-cre ROSA HuRfl/fl, in which HuR is ablated prior to T cell activation. This resulted in profound suppression of Th2 but not Th1 cytokines, under in vitro conditions. We used the ova challenge model of airway inflammation to study antigen presentation and in vivo cytokine regulation. HuR KO mice had completely abolished lung cellular inflammation to levels similar to non-immunized mice. This further resulted in significant reductions in IL-4, IL-13 secretion, and serum IgE levels. We also investigated effects of HuR ablation in Th17 differentiation. We show HuR regulates IL-17 expression by controlling transcript stability. HuR KO T cells have greatly reduced Th17 differentiation (66% reduction) and diminished onset and severity of inflammation in an EAE model. These data indicate that HuR is required for efficient CD4+ Th2 and Th17 differentiation both in vitro and in vivo but not Th1. These findings reveal nuanced control by HuR upon different CD4+ T cell subsets.

Optimization of mRNA design for protein expression in the crustacean Daphnia magna

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3' UTR failed to induce fluorescence. To assess reporter expression, the length of the 3' UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3' end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3' UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3' UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3' UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.

The Antiangiogenic Insulin Receptor Substrate-1 Antisense Oligonucleotide Aganirsen Impairs AU-Rich mRNA Stability by Reducing 14-3-3β–Tristetraprolin Protein Complex, Reducing Inflammation and Psoriatic Lesion Size in Patients

Increased inflammation and aberrant angiogenesis underlie psoriasis. Here, we report that the inhibition of insulin receptor substrate-1 (IRS-1) expression with aganirsen resulted in a dose-dependent reduction (P < 0.0001) in IRS-1 protein in the cytoplasm, while IRS-1 protein remained quantitatively unchanged in the perinuclear environment. Aganirsen induced a dose-dependent increase in serine-phosphorylated IRS-1 in the soluble perinuclear-nuclear fraction, inducing IRS-1-14-3-3β protein association (P < 0.001), thereby impairing 14-3-3β-tristetraprolin protein complex and AU-rich mRNA's stability (P < 0.001). Accordingly, aganirsen inhibited (P < 0.001) in vitro the expression of interleukin-8 (IL-8), IL-12, IL-22, and tumor necrosis factor alpha (TNFα), four inflammatory mediators containing mRNA with AU-rich regions. To demonstrate the clinical relevance of this pathway, we tested the efficacy of aganirsen by topical application in a pilot, double-blind, randomized, dose-ranging study in 12 psoriatic human patients. After 6 weeks of treatment, least square mean differences with placebo were -38.9% (95% confidence interval, -75.8 to -2.0%) and -37.4% (-74.3 to -0.5%) at the doses of 0.86 and 1.72 mg/g, respectively. Lesion size reduction was associated with reduced expression of IRS-1 (P < 0.01), TNFα (P < 0.0001), and vascular endothelial growth factor (P < 0.01); reduced keratinocyte proliferation (P < 0.01); and the restoration (P < 0.02) of normal levels of infiltrating CD4(+) and CD3(+) lymphocytes in psoriatic skin lesions. These results suggest that aganirsen is a first-in-class of a new generation of antiangiogenic medicines combining anti-inflammatory activities. Aganirsen-induced downregulation of inflammatory mediators characterized by AU-rich mRNA likely underlies its beneficial clinical outcome in psoriasis. These results justify further large-scale clinical studies to establish the dose of aganirsen and its long-term efficacy in psoriasis.

Role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of Dr+Escherichia coli receptor protein decay accelerating factor (DAF or CD55) by nitric oxide

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr(+) ). The epithelial invasion of Dr(+) E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by downregulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5'-untranslated region and mapped the NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5'-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3'-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to the AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF.

The ribosome binding site of a mini‐ORF protects a T3SS mRNA from degradation by RNase E

Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.

3' Untranslated region-dependent degradation of the aceA mRNA, encoding the glyoxylate cycle enzyme isocitrate lyase, by RNase E/G in Corynebacterium glutamicum.

We previously reported that the Corynebacterium glutamicum RNase E/G encoded by the rneG gene (NCgl2281) is required for the 5' maturation of 5S rRNA. In the search for the intracellular target RNAs of RNase E/G other than the 5S rRNA precursor, we detected that the amount of isocitrate lyase, an enzyme of the glyoxylate cycle, increased in rneG knockout mutant cells grown on sodium acetate as the sole carbon source. Rifampin chase experiments showed that the half-life of the aceA mRNA was about 4 times longer in the rneG knockout mutant than in the wild type. Quantitative real-time PCR analysis also confirmed that the level of aceA mRNA was approximately 3-fold higher in the rneG knockout mutant strain than in the wild type. Such differences were not observed in other mRNAs encoding enzymes involved in acetate metabolism. Analysis by 3' rapid amplification of cDNA ends suggested that RNase E/G cleaves the aceA mRNA at a single-stranded AU-rich region in the 3' untranslated region (3'-UTR). The lacZ fusion assay showed that the 3'-UTR rendered lacZ mRNA RNase E/G dependent. These findings indicate that RNase E/G is a novel regulator of the glyoxylate cycle in C. glutamicum.

Albumin 3′untranslated region facilitates increased recombinant protein production from Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are used for recombinant protein production in the pharmaceutical industry but there is a need to improve expression levels. In the present work experiments were carried out to test the effectiveness of different 3'untranslated regions (3'UTRs) in promoting production of a naturally secreted luciferase. Seamless cloning was used to produce expression vectors in which Gaussia princeps luciferase coding sequences were linked to the human albumin, immunoglobulin or chymotrypsinogen 3'UTR. Stably transfected CHO cells expressing these constructs were selected. Luciferase activity in the culture medium was increased 2-3-fold by replacing the endogenous 3'UTR with the albumin 3'UTR and decreased by replacement with immunoglobulin or chymotrypsinogen 3'UTR. Replacement of the native 3'UTR with the albumin 3'UTR led to a 10-fold increase in luciferase mRNA levels. Deletion analysis of the albumin 3'UTR showed that loss of nucleotides 1-50, which removed an AU-rich complex stem loop region, caused significant reductions in both luciferase protein expression and luciferase mRNA levels. The results suggest that recombinant protein expression and yield could be improved by the careful selection of appropriate 3'UTR sequences.

Evidence for cell cycle suppression and microRNA regulation of cyclin D1 during anoxia exposure in turtles

The red-eared slider turtle (Trachemys scripta elegans) has a well-developed natural tolerance for oxygen deprivation that derives from biochemical adaptations, including anoxia-induced suppression of metabolic rate. We hypothesized that mechanisms that suppress ATP-expensive cell cycle activity would contribute significantly to establishing the hypometabolic state during anaerobiosis. Cyclin D1 is a critical regulator of the G1 phase of the cell cycle and is regarded as key to initiating cell proliferation. The relative protein expression of cyclin D1 was analyzed in both whole-cell and nuclear fractions of liver, kidney and skeletal muscle from turtles exposed to 5 or 20 h of submergence anoxia. Expression of cyclin D1 in both total and nuclear fractions decreased significantly under anoxia in liver and kidney as compared with aerobic controls, but no significant change occurred in muscle. The relative phosphorylation state of cyclin D1 (threonine 286) was also unchanged during anoxia in all tissues. Since phosphorylation of threonine 286 is necessary for proteasomal degradation of cyclin D1, this implies that an alternative mechanism is responsible for cyclin D1 suppression in anoxia. Levels of cyclin D1 mRNA transcripts did not change under anoxia in any tissue, so a post-transcriptional method of regulation was implicated. Analysis of the 3’UTR of cyclin D1 showed the presence of both an AU-rich region and a conserved binding site for microRNA-16-1 and microRNA-15a. Levels of both microRNAs increased in liver and kidney (but not in muscle) under anoxic conditions, implicating microRNA inhibition of mRNA translation as the mechanism underlying the suppression of cyclin D1 protein levels in the anoxic turtle.

Noncanonical repression of translation initiation through small RNA recruitment of the RNA chaperone Hfq

The RNA chaperone Hfq is mostly known to help small regulatory RNAs (sRNAs) interact with target mRNAs to block initiating ribosomes. In this model, whereas the sRNA is directly competing with initiating 30S ribosomal subunits, Hfq plays only an indirect role, allowing optimal sRNA-mRNA pairing. Here we report that Hfq is recruited by a sRNA, Spot42, to bind to a precise AU-rich region in the vicinity of the translation initiation region (TIR) of sdhC mRNA and competes directly with 30S ribosomal subunits. We show that the sRNA Spot42 binds sdhC too far upstream of the TIR to directly repress translation initiation in vitro and in vivo. Contrary to the canonical model of sRNA regulation, this suggests a new mechanism where Hfq is directly involved in the translational repression of the target mRNA and where the sRNA acts only as a recruitment factor.

Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3'UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3'UTR that may act as regulatory sequences. Analysis of ODC 3'UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

Genome-Wide Assessment of AU-Rich Elements by the AREScore Algorithm

In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3′ untranslated region (UTR) of many short-lived mRNAs. AREs cause mRNAs to be degraded rapidly and thereby suppress gene expression at the posttranscriptional level. Based on the number of AUUUA pentamers, their proximity, and surrounding AU-rich regions, we generated an algorithm termed AREScore that identifies AREs and provides a numerical assessment of their strength. By analyzing the AREScore distribution in the transcriptomes of 14 metazoan species, we provide evidence that AREs were selected for in several vertebrates and Drosophila melanogaster. We then measured mRNA expression levels genome-wide to address the importance of AREs in SL2 cells derived from D. melanogaster hemocytes. Tis11, a zinc finger RNA–binding protein homologous to mammalian tristetraprolin, was found to target ARE–containing reporter mRNAs for rapid degradation in SL2 cells. Drosophila mRNAs whose expression is elevated upon knock down of Tis11 were found to have higher AREScores. Moreover high AREScores correlate with reduced mRNA expression levels on a genome-wide scale. The precise measurement of degradation rates for 26 Drosophila mRNAs revealed that the AREScore is a very good predictor of short-lived mRNAs. Taken together, this study introduces AREScore as a simple tool to identify ARE–containing mRNAs and provides compelling evidence that AREs are widespread regulatory elements in Drosophila.

Stabilization of cGMP-dependent protein kinase G (PKG) expression in vascular smooth muscle cells: contribution of 3'UTR of its mRNA

The type-I cGMP-dependent protein kinase (PKG-I) expression regulation is not yet completely understood. In this study, we examined the role of 3'-untranslated region (3'UTR)-PKG-I messenger RNA (mRNA) in the control of PKG-I expression in vascular smooth muscle cells (VSMCs). Using a 3'-rapid amplification of cDNA ends (RACE) for the amplification of complementary DNA (cDNA) ends, we generated and cloned a 1.2-kb-3'UTR mRNA PKG-I in pGL3 control vector downstream of the luciferase reporter gene. Serial deletions and functional studies revealed that among the deleted constructs, only the 1.2-kb-3'UTR PKG-I mRNA possesses the highest activity in transfected VSMC. Kinetic luciferase assays in the presence of actinomycin D showed that this construct stabilizes luciferase activity compared to the control vector. Sequence analysis of 3'UTR-PKG-I mRNA revealed the existence of four AU-rich regions (AU1 through AU4) in addition to a potential poly(A) site. Different riboprobes were generated either by 5'-end-labelling of designed ribonucleotides, containing individual AU-rich regions or by in vitro transcription assay using cloned 1.2-kb cDNA as a template. RNA-electrophoretic mobility shift assay (EMSA) and ultra-violet cross-linking (UV-CL) assays showed that AU1, AU3, AU4 and 1.2-kb probes were able to retard cytosolic and nuclear proteins. Taken together, these data suggest that PKG-I expression is subjected to post-transcriptional regulation in VSMC through the 3'UTR of its mRNA.

Structural and optical studies of Au doped titanium oxide films

Thin films of TiO 2 were doped with Au by ion implantation and in situ during the deposition. The films were grown by reactive magnetron sputtering and deposited in silicon and glass substrates at a temperature around 150 °C. The undoped films were implanted with Au fluences in the range of 5 × 10 15 Au/cm 2–1 × 10 17 Au/cm 2 with a energy of 150 keV. At a fluence of 5 × 10 16 Au/cm 2 the formation of Au nanoclusters in the films is observed during the implantation at room temperature. The clustering process starts to occur during the implantation where XRD estimates the presence of 3–5 nm precipitates. After annealing in a reducing atmosphere, the small precipitates coalesce into larger ones following an Ostwald ripening mechanism. In situ XRD studies reveal that Au atoms start to coalesce at 350 °C, reaching the precipitates dimensions larger than 40 nm at 600 °C. Annealing above 700 °C promotes drastic changes in the Au profile of in situ doped films with the formation of two Au rich regions at the interface and surface respectively. The optical properties reveal the presence of a broad band centered at 550 nm related to the plasmon resonance of gold particles visible in AFM maps.